2.2.8 研究 | 2021-多组学分析肺结核队列的记忆T细胞状态

刘小泽写于2021.5.25

速览

• 题目：Multimodally profiling memory T cells from a tuberculosis cohort identifies cell state associations with demographics, environment and disease

• 作者：哈佛医学院 Soumya Raychaudhuri 团队

  首先感觉他们实验室主页做的很不错：https://immunogenomics.hms.harvard.edu/lab

• 链接：https://www.nature.com/articles/s41590-021-00933-1

• 发表在：Nature Immunology （2021-05-24）

• 数据是公开的：GSE158769 ，采用CITE-seq

  Platforms    GPL11154    Illumina HiSeq 2000 (Homo sapiens)
  Samples      GSM4810298    Memory_T_cell_CITE-seq
  
  GSE158769_exprs_norm.tsv.gz    3.0 Gb    (ftp)(http)    TSV
  GSE158769_exprs_raw.tsv.gz    747.0 Mb    (ftp)(http)    TSV
  GSE158769_meta_data.txt.gz    19.8 Mb    (ftp)(http)    TXT

  Genotype data：phs002025.v1.p1

• 分析代码：https://github.com/immunogenomics/TB_Tcell_CITEseq

• 还发开了一个网页：https://immunogenomics.io/tbru/

摘要

• 分析了来自秘鲁的259个肺结核（tuberculosis , TB)病人的500,089个记忆T细胞，得到31种细胞状态

• 在排除年龄、性别、季节、遗传等因素影响后，发现了一种多能型type 17 helper T (TH17) 细胞状态，在结核杆菌发展成肺结核病的过程中丰度和功能都有所下降

方法

数据准备

• PBMC sample preparation

• Flow cytometry of total PBMCs

• CITE-seq of isolated memory T cells

• Bulk RNA sequencing

• Genotyping and genetic data processing

数据分析

• scRNA比对+定量

• scRNA数据拆分

• scRNA数据质控

• 降维

• 聚类+注释

• bulk RNA-seq 比对+定量

• 利用MASC(modeling of associations of single cells )评价不同细胞状态与疾病的关系

先用CITE-seq测了>500,000个T细胞

• 在秘鲁首都利马进行了一次大型的流行病学调查（n= 18,544），从其中招募了264个人，采样了外周血单核细胞进行CITE-seq测定，同时还加入了基因表型数据和蛋白数据

• 图b是进行了6次QC，每一次过滤掉一些细胞，最后得到了500K

• 大部分样本的细胞数量在2k左右

得到记忆T细胞的31种不同状态

利用canonical correlation analysis (CCA) 进行降维，选择top20 canonical variates (CVs)进行批次校正和聚类，根据基因和蛋白marker得到31个细胞状态

• 23/31 were CD4+; five were CD8+; one (C-24) was a mixture; Two clusters (C-30 and C-31) were CD4−CD8−

• C-20： contained a subset of CD4−CD8− and CD8+ cells expressing innate-like T cell markers, including ZBTB16 and CD161 and CD26 surface proteins

• one central memory cluster (C-25) and distinct GZMK+ (C-28) and GZMB+ (C-29) effector subsets, reflecting different cytotoxicities

• high expression of HLA-DR and CD38 surface protein and proliferation-associated MKI67 (C-15 and C-27) represent chronically activated cells

• TH17 cells (C-12, CCR6+ and RORC)

• type 1 helper T cells (TH1; C-17, CXCR3+ and IFNG and TBX21)

• heterogeneous continuum of intermediate TH1/TH17 states (C-13, C-16 and C-19) with varying degrees of CXCR3, CCR6, CCR5 and CD161 surface protein expression and RORC and TBX21 expression

• CD161+ subset of type 2 helper T (TH2) cells (C-14), described as pathogenic, with higher expression of allergy-associated HPGDS and IL17RB

• a subset of FOXP3+ Treg cells (C-5) expressing higher CCR6 surface protein and CTLA4 and RORC than other Treg cells

记忆T细胞的状态因人口特征和环境而异

发现： 年龄因素的影响最大；性别因素与T细胞状态也高度相关； type 2 helper T (TH2) 细胞状态在冬天（季节因素）搜集的样本中最为丰富

之后发现一个感兴趣的C-12状态

探索每个细胞状态和肺结核病情进展的关系，同时校正其他可能存在影响的因素（年龄、性别、季节、遗传以及其他技术因素），发现：

病人在肺结核发展时期，C-12显著降低了20%，并且在年龄因素和男性因素下也会下降，而季节因素（冬季）会上升

又找到了C-12 surface markers：CD4+CD26+CD161+CCR6+ ，这些marker已经被证明了以下功能：

• IL-17-producing TH17 state

• CD26 is a co-stimulatory molecule that promotes cytotoxicity

• CD161 is associated with innate-like function

• CCR6 is a homing marker that directs migration to inflamed sites

那么C-12的下降是疾病的原因还是结果呢？

利用外部数据进行验证，发现这种变化可能早于疾病发展

• predicted C-12 abundance was 9% lower in cases before progression compared to latent controls